1. Field of the Invention
The present invention generally relates to plant genetics. More specifically, the invention relates to genes involved in the biosynthesis of anthocyanins, proanthocyanidins, and tannins in alfalfa (Medicago sp.), and methods for use thereof.
2. Description of the Related Art
Proanthocyanidins (PAs), also known as condensed tannins (CTs), are polymers of flavonoid (flavan-3-ol) units. Their name reflects the fact that, on acid hydrolysis, the extension units are converted to colored anthocyanidins, and this forms the basis of the classical assay for these compounds (Porter 1989). Anthocyanins and proanthocyanidins are found in many plant species. Anthocyanins contribute to the coloration of plant tissues, may act as attractants to pollinators (Schemske and Bradshaw, 1999), and provide UV protection (Reddy et al., 1994). PAs provide plants with protection against insects, herbivores and fungal infection (Schultz and Baldwin, 1982; Bending and Read, 1996). Recently, realization of the beneficial qualities of dietary PAs for human health has increased the interest in these compounds (Bagchi et al., 2000; Dufresne and Farnworth, 2001). Simple monomeric and/or oligomeric PAs have been shown to possess anticancer, antioxidant and antimicrobial activities (Dixon et al., 2005).
Modest amounts of PAs in forages promote increased dietary protein nitrogen utilization and reduced occurrence of pasture bloat in ruminant animals such as cattle and sheep (Li et al., 1996; Aerts et al., 1999; Barry and McNabb, 1999). Pasture bloat occurs in ruminants when they are fed with a high protein diet such as alfalfa (lucerne; Medicago sativa) or clover (Trifolium spp), species that lack PAs in their aerial portions. The combination of excessive protein and methane released in the rumen from fermentation of the forage results in formation of a thick foam leading to bloating which, in severe cases, can be fatal. To combat pasture bloat, a common practice is to supplement the ruminant diet with surfactants, which break down the protein foams (Hall et al., 1994). Another remedy involves mixing high protein forage with forage known to contain moderate levels of PAs (Li et al., 1996). Both of these are costly options for the farmer or rancher, resulting in a reluctance to take advantage of the otherwise excellent nutritional qualities of alfalfa and clovers.
The building blocks of most PAs are (+)-catechin and (−)-epicatechin. (−)-Epicatechin has 2,3-cis stereochemistry and (+)-catechin has 2,3-trans-stereochemistry. These stereochemical differences are of major importance in PA biosynthesis, since all chiral intermediates in the flavonoid pathway up to and including leucoanthocyanidin are of the 2,3-trans stereochemistry, raising important questions about the origin of the 2,3-cis stereochemistry of (−)-epicatechin, the commonest extension unit in proanthocyanidins (Foo and Porter 1980). The most common anthocyanidins produced are cyanidin (leading to procyanidins) and delphinidin (leading to prodelphinidins). PAs may contain from 2 to 50 or more flavonoid units. PA polymers have complex structures because of variations in the flavonoid units and the sites for interflavan bonds. Depending on their chemical structure and degree of polymerization, PAs may or may not be soluble in aqueous organic solvents.
PAs are attracting increasing attention due to their ability to affect the nutritional quality of human and animal food (Bagchi et al., 2000; Barry and McNabb, 1999; Morris and Robbins, 1997). In addition, PAs and anthocyanins from various plants have beneficial effects on cardiac health and immune responses (Pataki et al., 2002; Foo et al., 2000; Lin et al., 2002), and to prevent macular degeneration (e.g. Brevetti et al., 1989; Lee et al., 2005). PAs can reversibly bind to proteins and reduce their degradation rate. The presence of moderate amounts of PAs in forage crops reduces the initial rate of microbial digestion of the protein component of forage material in the rumen. The protein-PA complexes then pass to the abomasum where they dissociate at the lower pH, providing “by-pass protein” for utilization by the animal and consequent enhancement of milk and wool production and live weight gain (Barry and McNabb, 1999; Tanner et al., 1995).
In addition, low concentrations of PA can help counter intestinal parasites in lambs, and confer bloat safety, presumably by interacting with both leaf protein and microbial enzymes such that the rate of protein degradation in the rumen is reduced (Aerts et al 1999). These properties of PAs underscore the potential importance of methods of engineering PA synthesis in crops, including forage crops in particular.
In addition, it has been shown that the presence of PAs in forage crops significantly reduces emission of the greenhouse gas methane by farm animals. Farm animals have been shown to produce large amounts of methane (˜80 kg/yr/cow). Furthermore, PAs also preserve proteins during the ensiling process, increasing the feed value of silage and reducing the amount of nitrogen that is lost to the environment as feedlot waste (Albrecht and Muck, 1991; Reed, 1995). In laboratory studies, treatment of feed proteins with modest amounts of PAs (around 2-4% of dry matter) reduced proteolysis during both ensiling and rumen fermentation. In studies performed with sheep in New Zealand, increasing dietary PAs from trace amounts to 4% of dry matter increased by-pass protein, and a diet containing only 2% PAs strongly increased absorption of essential amino acids by the small intestine by up to 60% (Douglas et al. 1999).
An attractive alternative for forage improvement lies in genetically transferring the capability to synthesize PAs to non PA-accumulators (e.g. see WO 06/010096 or US Publication 2006/0123508). Since the precursors for PAs are the same as those for the production of anthocyanins, one approach is to transform plants with a transcription factor which, when ectopically expressed, induces anthocyanin production. Co-expression of one or more PA-specific biosynthetic enzymes such as anthocyanidin reductase (ANR), which converts cyanidin to the flavan-3-ol (−)-epicatechin, a building block of PAs (FIGS. 1-2) (Dixon et al., 2005), may then lead to PA accumulation (Xie et al., 2006).
However, there are several technical problems with this approach. First, apart from enzymes converting anthocyanin pathway precursors to catechin and epicatechin (US Publication 20040191787; Tanner et al., 2003; Xie et al., 2003; US Publication 20060123508), two potential transporters (Debeaujon et al., 2001; Kitamura et al., 2004), and an oxidase that likely acts on polymerized products (Pourcel et al., 2005), little is known of the proteins necessary for polymerization of tannins and their ultimate accumulation in vacuoles or cell walls (Dixon et al., 2005; Xie and Dixon, 2005). Second, transcription factors controlling anthocyanin production appear to be species-specific. Whereas the Arabidopsis thaliana producer of anthocyanin pigmentation (AtPAP1) MYB transcription factor (GenBank Accession AF325123) effectively induces anthocyanin production in Arabidopsis and tobacco (Borevitz et al., 2000), it does not function in alfalfa or white clover (see below). Similarly, expression of the maize Lc gene in alfalfa only resulted in anthocyanin production if the plants were exposed to strong abiotic stress (Ray et al., 2003). Expression of maize Lc Myc in conjunction with other transcription factors in Arabidopsis could lead to premature necrosis and death of the plants (Sharma and Dixon, 2005). Finally, even if anthocyanin production and downstream enzymes (for PA synthesis) are expressed, tannins have not necessarily accumulated, as seen in Arabidopsis expressing multiple flavonoid-pathway transcription factors (Sharma and Dixon, 2005).
The foregoing studies have provided a further understanding of the mechanisms and manipulation of plant secondary metabolism. However, the prior art has failed to provide techniques for the application of this understanding to the creation of plants having valuable new characteristics. What are thus needed are practical techniques for the production of novel plants with improved phenotypes and methods for the use thereof. Such techniques may allow the creation and use of plants with improved nutritional quality, thereby benefiting both human and animal health and representing a substantial benefit in the art.